Apparatus and method for mixing a film of fluid

ABSTRACT

A method and apparatus is provided for mixing a film of fluid, particularly a film of chemical, biochemical, or biological fluids undergoing a reaction. The apparatus comprises a means for nucleating a bubble using a discrete heat source, such as a resistor, and moving the bubble in the fluid by creating a temperature gradient, thereby mixing the fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/782,542 filed Feb. 12, 2001 (U.S. Pat. No. 6,513,968) which is acontinuation of U.S. patent application Ser. No. 09/137,963 filed Aug.21, 1998 (U.S. Pat. No. 6,186,659). Priority is claimed from both of theforegoing applications under 35 U.S.C. 120 and those applications areincorporated herein by reference.

TECHNICAL FIELD

This invention relates to mixing fluids, and more particularly relatesto an apparatus and method for mixing a small quantity of fluid that ispresent as a film on a solid substrate.

BACKGROUND

Methods for mixing relatively large volumes of fluids usually utilizeconventional mixing devices that mix the fluids by shaking thecontainer, by a rapid mechanical up and down motion, or by the use of arocking motion that tilts the container filled with the fluids in a backand forth motion. The conventional mixing methods normally cannot beutilized for methods involving thin films of fluid because the capillarystrength of the containment system often significantly exceeds theforces generated by shaking or rocking, thereby preventing or minimizingfluid motion in the film. The problem is illustrated by a surfacechemistry reaction where the surface is large and the available fluidsample is very small. The fluid when spread across the surface resultsin a thin film of fluid that may have a thickness of a few microns to afew millimeters. In such situations, the fluid may not adequatelycontact the entire reactive surface or the reactive compounds in thefluid may be very dilute thereby resulting in a reaction that is limitedby the rate of diffusion through the fluid. Thus, inadequate mixing canadversely affect the sensitivity or specificity of the reaction, therate of reaction, the extent of reaction, the homogeneity, or thepercent yield.

Inadequate mixing is a particular problem in chemical and biologicalassays where very small samples of chemical, biochemical, or biologicalfluids are typically reacted. For example, the ability to clone andsynthesize nucleotide sequences has led to the development of a numberof techniques for disease diagnosis and genetic analysis. Geneticanalysis, including correlation of genotypes and phenotypes, contributesto the information necessary to reveal the changes in genes which conferdisease. New methods of diagnosis of diseases, such as AIDS, cancer,sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, andthe like, rely on the detection of mutations present in certainnucleotide sequences. Many of these techniques generally involvehybridization between a target nucleotide sequence and a complementaryprobe, offering a convenient and reliable means for the isolation,identification, and analysis of nucleotides.

One typical method involves hybridization with either target or probenucleotide sequences immobilized on a solid support. The targets orprobes are usually immobilized on a solid support having a surface areaof typically less than a few square centimeters. The solid support istypically a glass or fused silica slide which has been treated tofacilitate attachment of either the targets or probes. The mobile phasecontaining reactants that react with the attached targets or probes isplaced on the support, covered with another slide, and placed in anenvironmentally controlled chamber such as an incubator. Normally, thereactants in the mobile phase diffuse through the liquid to theinterface where the complementary probes or targets are immobilized, anda reaction, such as hybridization reaction, then occurs. Preferably, themobile phase reactants are labeled with a detectable tag, such as afluorescent tag, so that the hybrid could be identified. Thehybridization reaction typically takes place over a time period that canbe many hours.

Problems are often encountered in conducting chemical or biologicalassays, including use of arrays, with poor hybridization kinetics andefficiency or reaction specificity and sensitivity, since diffusion isthe only means of moving the reactants in the mobile phase to theinterface or surface containing the immobilized reactants.Alternatively, the fluid sample must be removed from the reactionchamber, mixed in separate chambers external to the reaction chamber,and then reintroduced into the reaction chamber. Valuable fluid sampleis wasted or lost in the separate external chambers required in suchmixing process.

A method and apparatus for mixing a thin film of fluid, particularly achemical, biochemical, or biological fluid undergoing a reaction, isdescribed in a copending application U.S. Ser. No. 08/889763. Theapplication describes a thin film of fluid between two opposing surfacesthat is mixed by moving one surface relative to the other.

The present invention describes an apparatus and method for mixing of afilm of fluid via nucleation of bubbles within the film. The use ofbubbles for mixing large volumes of liquids is well known. For example,U.S. Pat. No. 5,443,985 to Lu et al. and U.S. Pat. No. 5,605,653 toDeVos describe the mixing and aeration of large volumes of liquid, suchas a culture medium in a cell culture bioreactor by introducingextraneous gas at the bottom of the reactor thereby creating bubblesthat travel upwards, thus mixing the liquid medium. In another context,U.S. Pat. No. 5,275,787 to Yaguchi et al. describes the use of thermalenergy to generate a bubble that is then used to discharge a sampleliquid containing individual particles. The generation of the bubble andits use as an optical switching element for devices that have uses intelecommunication systems and data communication systems is described inU.S. Pat. No. 5,699,462 to Fouquet et al. and U.S. Pat. No. 4,988,157 toJackel et al.

SUMMARY OF THE INVENTION

The invention, in one embodiment, is an apparatus for mixing a film offluid, particularly a chemical, biochemical, or biological fluid whichtypically comprises a reaction mixture, the apparatus comprising a firstsubstrate having an inner surface and a substantially parallel secondsubstrate having an inner surface that defines a closed chambertherebetween. The closed chamber is adapted to retain a quantity offluid so that the fluid is in contact with both inner surfaces. Inaddition, the apparatus comprises a means for nucleating bubbles in thefluid comprising discrete heat sources for creating individual bubblesat selected locations within the apparatus such that as each bubble isnucleated and dispelled, the fluid is displaced resulting in mixing, anda means for moving the bubbles in the fluid. The inner surface of one orboth of the substrates is functionalized with reactive moieties that canreact with the components contained in the fluid. The bubbles arenucleated by using discrete heat sources, such as resistors arranged ina predetermined pattern adjacent to the inner surface of the substrate.

In another embodiment, only the first substrate has the means fornucleating bubbles comprising discrete heat sources adjacent to theinner surface and is functionalized with reactive moieties.

The invention also provides a method for mixing a film of fluidcomprising providing a first substrate and a substantially parallelsecond substrate having inner surfaces that define a closed chambertherebetween. The chamber is adapted to retain a quantity of fluid sothat the fluid is in contact with both inner surfaces. The methodfurther comprises introducing a fluid containing a plurality ofcomponents into the closed chamber so as to provide a film of fluidtherein, nucleating a bubble within the fluid film, whereby, as eachbubble that is nucleated is dispelled, the fluid is displaced resultingin mixing, and a means for moving a bubble in the film of fluid. Thebubbles may be moved in the film of fluid by creating a temperaturegradient along which the bubbles move.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an apparatus of the present inventionwherein the first substrate 10 is substantially parallel to the secondsubstrate 11 with a seal 15 in between.

FIG. 2 is a cross-sectional view of the apparatus shown in FIG. 1,wherein discrete resistors 13 are adjacent to the inner surface 12 ofthe first substrate 10 with a seal 15 in between the first 10 and thesecond 11 substrates.

FIG. 3 is a edge on view of an apparatus of the present invention,wherein the first substrate 10 and the second substrate 11 aresubstantially parallel with a seal 15 in between.

FIG. 4 is a cross-sectional view of the apparatus shown in FIG. 3,illustrating the plurality of chemical, biochemical, or biologicalmoieties 14 attached to the inner surface 12 of the second substrate 11and discrete resistors 13 adjacent to the inner surface of the firstsubstrate 10.

DETAILED DESCRIPTION OF THE INVENTION

Overview and Definitions:

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,reagents, process steps, or equipment, as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “bubble” as used herein refers to a small ball of gas in afluid wherein the gas originates from fluid vapor (a vapor bubble) ordissolved gas (a gas bubble) in the fluid. The word “bubble” used aloneencompasses both a gas bubble and a vapor bubble.

The term “fluid” as used herein refers to a material that can flow suchas a liquid, or a semisolid.

The term “functionalization” as used herein relates to modification of asolid substrate to provide a plurality of functional groups on thesubstrate surface. By a “functionalized surface” as used herein is meanta substrate surface that has been modified so that a plurality offunctional groups are present thereon.

The term “monomer” as used herein refers to a chemical entity that canbe covalently linked to one or more other such entities to form anoligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptides, and the like. In general, the monomers used inconjunction with the present invention have first and second sites(e.g., C-termini and N-termini, or 5′ and 3′ sites) suitable for bindingto other like monomers by means of standard chemical reactions (e.g.,condensation, nucleophilic displacement of a leaving group, or thelike), and a diverse element which distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid sidechain, a nucleotide base, etc.). The initial substrate-bound monomer isgenerally used as a building-block in a multi-step synthesis procedureto form a complete ligand, such as in the synthesis of oligonucleotides,oligopeptides, and the like.

The term “oligomer” is used herein to indicate a chemical entity thatcontains a plurality of monomers. As used herein, the terms “oligomer”and “polymer” are used interchangeably, as it is generally, although notnecessarily, smaller “polymers” that are prepared using thefunctionalized substrates of the invention, particularly in conjunctionwith combinatorial chemistry techniques. Examples of oligomers andpolymers include polydeoxyribonucleotides, polyribonucleotides, otherpolynucleotides which are—or C-glycosides of a purine or pyrimidinebase, polypeptides, polysaccharides, and other chemical entities thatcontain repeating units of like chemical structure. In the practice ofthe instant invention, oligomers will generally comprise about 2-50monomers, preferably about 15-30 monomers.

The term “ligand” as used herein refers to a moiety that is capable ofcovalently or otherwise chemically binding a compound of interest.Typically, when the present substrates are used in solid phasesynthesis, they are used so that “ligands” are synthesized thereon.These solid-supported ligands can then be used in screening orseparation processes, or the like, to bind a component of interest in asample. The term “ligand” in the context of the invention may or may notbe an “oligomer” as defined above. However, the term “ligand” as usedherein may also refer to a compound that is not synthesized on the novelfunctionalized substrate, but that is “pre-synthesized” or obtainedcommercially, and then attached to the substrate.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties which contain not only the known purine and pyrimidine bases,but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, or other heterocycles. In addition, the terms“nucleoside” and “nucleotide” include those moieties that contain notonly conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like. As used herein, the term “amino acid” isintended to include not only the L-, D- and nonchiral forms of naturallyoccurring amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine), but also modified amino acids, amino acidanalogs, and other chemical compounds which can be incorporated inconventional oligopeptide synthesis, e.g., 4-nitrophenylalanine,isoglutamic acid, isoglutamine, ε-nicotinoyl-lysine, isonipecotic acid,tetrahydroisoquinoleic acid, α-aminoisobutyric acid, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, 4-aminobutyric acid, and the like.

Accordingly, the invention in a first embodiment is directed to anapparatus, shown in FIGS. 1 and 2, comprising a first substrate 10having an inner surface 12 that can retain a film of fluid and a meansof nucleating bubbles in the film of fluid. The substrate may becomposed of any material that is compatible with the fluids with whichthe surface comes in contact with, such as, for example, glass, silicon,fused silica, metal, ceramic, plastic, or other polymers such as nylon.The substrate may be rigid or flexible, and may define a shape that issubstantially planar in the shape of a circle, an ellipse, a square, arectangle, a triangle, or any other convenient substantially planarshape. The surface area of the inner surface that comes in contact withthe fluid is on the order of about 10 mm² to about 20 cm², morepreferably from about 100 mm² to about 10 cm², and most preferably about200 mm² to about 10 cm². The volume of the fluid that can be retained bythe inner surface is less than about 300 μl, more preferably less thanabout 150 μl, even more preferably less than about 50 μl.

The inner surface of the substrate has means for nucleating bubbles inthe film of fluid, comprising discrete heat sources, radiofrequencysources, microwave sources, light sources, or mechanical sources. In thepreferred embodiment, the means for nucleating bubbles comprise discreteheat sources, preferably resistors 13. The resistors are adjacent to theinner surface of the substrate and in thermal communication with thefilm of fluid retained thereon. The resistors are electrically connectedto a control circuit that controls the voltages or currents applied tothe resistors to nucleate the bubble. Bubbles are thus nucleated byheating the fluid in the local environment around the resistors.Subsequently, the control circuit may selectively reduce the electricalinput to the resistors to allow the bubble to dissipate or collapse.

The apparatus for mixing a film of fluids also has means for moving abubble through the fluid. In the preferred embodiment, as a heat sourceis turned off, a neighboring heat source may be turned on, therebycreating a temperature gradient in the fluid. The bubble will move alongthe temperature gradient from the region of lower temperature towardsthe region of higher temperature. The movement of the bubble along thetemperature gradient, caused by thermocapillary action, displaces thelocal fluid thereby mixing it. Thus, the control circuit can control thetemperature gradient. In addition, the control circuit can control theambient temperature of the film of fluid by supplying an identicalamount of voltage or current to nearly all the resistors at the sametime.

The fluid can be mixed locally by moving a bubble back and forth betweentwo neighboring heat sources. Alternatively, a plurality of bubbles canbe nucleated at a plurality of locations and moved independently of oneanother. In addition, a pre-fabricated pattern of heat sources can becreated such that the bubble is moved from one heat source to the nextalong the temperature gradient dictated by the pattern. The pattern ofthe heat sources can be linear, circular, rectangular, or any otherconvenient pattern. Moving the bubble along a circular pattern, forexample, induces a stirring motion in the film of fluid. Similarly, ifthe temperature gradient is linear, the bubble will move back and forthalong the line thereby inducing a back and forth motion in the fluid.

The inner surface of the substrate is functionalized with reactivemoieties to provide a plurality of functional groups on the substratesurface, typically hydroxyl, carboxylic, amido, or amino groups, thatare preferably bound to a ligand. The reactive moieties can becovalently bound or otherwise chemically bound to the ligand through avariety of methods known in the art. Optimally, the density of thechemical, biochemical, or biological ligands is greater than about 100per square micron, more preferably greater than about 1,000 per squaremicron, an even more preferably greater than about 5,000 per squaremicron. The ligands are selected from a monomeric species having atleast one reactive site, DNA, RNA, proteins, peptides, prions, reagents,and combinations, derivatives, and modifications thereof. See forexample, Kreiner (1996) “Rapid Genetic Sequence Analysis Using a DNAProbe Array System,” American Laboratory March: 39-43; Lipshutz et al.(1995) “Using Oligonucleotide Probe Arrays to Access Genetic Diversity,”BioTechniques 19: 442-447; Fodor et al. (1991) “Light-directed,Spatially Addressable Parallel Chemical Synthesis,” Science 252:767-772; Medlin (1995) “The Amazing Shrinking Laboratory,” EnvironmentalHealth Perspectives 103: 244-246; Southern et al. (1992) “Analyzing andcomparing Nucleic Acid Sequences by Hybridization to Arrays ofOligonucleotides: Evaluation Using Experimental Models,” Genomics 13:1008-1017; and Gacia et al. (1996) “Detection of Heterozygous Mutationsin BRCA1 using High Density Oligonucleotide Arrays and Two-colorFluorescence Analysis,” Nature Genetics 14: 441-447; and U.S. Pat. Nos.5,585,639; 5,601,980; and 5,551,487.

Optionally, a seal 15 can be attached to the outer periphery of thesubstrate, thus creating a chamber for the fluid with a definedthickness. The fluid is applied to an application site near the centerof the apparatus, and then distributed across the first surface to givethe film of fluid. Prior to the initiation of the mixing process, thefluid can be distributed across the inner surfaces of the substrate by,for example, rotating the apparatus, or by spreading with a cover slip.In addition, a second substrate 11, or a simple glass cover slip isplaced on top of the seal where the substantially parallel substratesdefine a closed chamber 16. The closed chamber may be a micron toseveral millimeters in thickness, preferably from about 5 microns toabout 100 microns in thickness.

Alternatively, two or more different fluids, each containing a reactivemoiety, can be introduced into the closed chamber and mixed tosubstantial homogeneity. This is advantageous when it is inconvenient ornot possible to attach the ligands to the reactive moiety on the innersurface of the solid substrate.

In addition, a dye, label, tag, reagent, or derivatives, modifications,or combinations thereof may be dried, lyophilized, or otherwise attachedto the inner surface of the substrate. The chemical, biochemical, orbiological fluid is introduced into the closed chamber, and the mixingprocess initiated to allow the desired reaction or labeling to occur.The reaction can be monitored after the mixing process is completed orinterspersed with the mixing process if necessary or desired. Thisprocess is particularly advantageous for fluids or reactions which areoptically dense and, therefore, must be analyzed in a very thin sectionor film by a spectrophotometer or other analytical means. The process isalso an advantage for specimens such as cellular suspensions or thinbiopsies which will be visually inspected in thin sections or films foraccurate count, diagnosis, or other analysis. This process is also anadvantage when the sample is particularly valuable or only available inminute quantities.

Electrical connection (not shown) is made to the resistors adjacent theinner surface of the substrate, and a bubble is nucleated at one or moreresistors. Turning off a resistor removes the heat and collapses thenucleated bubble, thereby displacing the liquid resulting in mixing.Preferably, as one resistor is turned off, if a neighboring resistor isactivated, a temperature gradient is created and the nucleated bubblemoves towards the hotter area resulting in a mixing action. The mixingaction is continued for the required period of time.

The apparatus having closed edges, shown in FIG. 3, may additionallyinclude a seal 15 between the two opposing inner surfaces. The seal canbe solid or flexible, and is fabricated from, for example, adhesives,rubber, plastic, glass, or metal. The apparatus preferably includes anopening in one of the substrates or in the seal for introducing fluidinto the closed chamber. The opening may be a port or other entrance.The fluid may be introduced by centrifugal means, pressure means, vacuummeans, positive displacement means, or other means known in the art.

The inner surface of first solid substrate 10 can have both the meansfor nucleating bubbles in the film of fluid and be functionalized withreactive moieties. Alternatively, as shown in FIG. 4 the inner surfaceof one of the substrates can have the means for nucleating bubbles 13 inthe film of fluid and the inner surface of the other substrate can befunctionalized with reactive moieties 14, wherein the fluid is retainedin the closed chamber 16. Finally, the inner surfaces of both substratescan have means for nucleating bubbles in the film of fluid and both canbe functionalized with reactive moieties that are bound to ligands whichare identical or preferably different.

The apparatus can be adapted for automated use, such as through the useof various controllers, computers, and the like. In addition, theapparatus can be adapted for use with multiple fluid chamberssimultaneously, where the temperature controlled environment is capableof containing and mixing multiple fluid chambers.

EXAMPLES Example 1 Hybridization to DNA Arrays

An array of DNA probes is constructed by attaching a plurality of knownprobes comprising oligomers, PCR product, or cDNA at specific locationson the inner surface of the substrate using techniques well known in theart. The substrate is a circular glass slide having a surface area ofabout 100 mm², and having resistors on the inner surface that areconnected to a electrical source. A rubber seal is placed around theouter edge of the substrate. About 50 μl of reactive fluid, comprising asample of mRNA, is placed on the inner surface of the solid substrate. Aglass slide is placed parallel to the first substrate and on top of theseal. The substrate is then rotated by a rotating mechanism, such as amotor, at an appropriate rotational speed in order to distribute thereactive fluid across the first surface of the substrate. The rotationalspeed may, for example, be in the range of a few hundred rpm to severalthousand rpm. Once the fluid is distributed over the surface of thesubstrate, the fluid may be heated to an optimum temperature for thereaction. For example, the fluid may be heated to 65° C. forhybridization studies where the surface probes are cDNA, or the fluidmay be heated to 37° C. where the surface probes are oligomers.

For mixing the fluid, voltage or current is applied to at least one ofthe resistors that nucleates a bubble in the fluid. Then, voltage orcurrent is turned off while voltage or current to the neighboringresistor is turned on. This creates a temperature gradient and thebubble is lured towards the hotter zone. The bubble is thus moved in acircular pattern, inducing a stirring motion in the film of fluid. Afterhybridization is complete, the sample is removed from the apparatus, theinner surface of the substrate is washed, and the apparatus is thenanalyzed to determine the quantity of mRNA that has hybridized to eachlocation. The entire process can be automated by use of computercontrol.

Example 2 Hybridization to RNA Arrays

An array of mRNA probes in a rectangular pattern is constructed atspecific locations on the inner surface of the solid substrate usingtechniques well known in the art. The substrate is a rectangular glassslide having a surface area of about 100 mm². A second substrate ofsilicon has heating sources that are resistors adjacent to the innersurface that are connected to a electrical source. The resistors arearranged in a square pattern. The two inner surfaces are held inparallel to each other in an opposing relationship, and then a glassseal is applied between the two substrates. Into the closed chamber thuscreated, about 50 μl of reactive fluid, comprising a sample of DNA, isplaced by positive pressure. The fluid is then heated to 45° C. byapplying a constant predetermined voltage to all the electrodes.

As in Example 1, additional voltage or current is applied to at leastone of the resistors that nucleates a bubble in the fluid. Then, voltageor current is returned to the base level while voltage or current to theneighboring resistor is increased. This creates a temperature gradientand the bubble is moved in a square pattern, inducing a stirring motionin the film of fluid. After hybridization is complete, the sample isremoved from the apparatus, the inner surface of the substrate iswashed, and the apparatus is then analyzed to determine the quantity ofDNA hybridized at each location.

Example 3 Solid Phase Synthesis of Polystyrene

Styrene is covalently bound via the aromatic ring to specific locationson the inner surface of the first substrate using techniques well knownin the art, such that the ethylene moiety remains reactive. The firstsubstrate is a circular silicon slide having a surface area of about 10cm², and having a plurality of resistors on the inner surface that areconnected to a electrical source The resistors are arranged in circularpattern. About 250 μl of reactive fluid, comprising a sample of styreneand 2,2′-azo-bis-isobutyronitrile as the free radical initiator, isplaced on the inner surface of the first solid substrate. The innersurface of a second substrate is held parallel to the inner surface ofthe first substrate, and a glass seal is then applied between the twosubstrates. The apparatus is rotated by a rotating mechanism, as inExample 1, to evenly distribute the fluid. The fluid is then heated to30° C. as in Example 2.

Additional voltage or current is applied to at least one of theresistors that nucleates a bubble in the fluid. Then, voltage or currentis returned to the base level while voltage or current to theneighboring resistor is applied. This creates a temperature gradient andthe bubble is moved in a back and forth pattern across the length of thesolid substrate. After reaction is complete, the sample is removed fromthe apparatus, the inner surface of the first substrate is washed, and afresh sample of fluid containing monomer suitable for polymerization isadded. The steps above are repeated until the desired length of thepolymer is obtained. The polymer chain is then dissociated from thefirst substrate to give free polystyrene.

It is to be understood that while the invention has been described inconjunction with preferred specific embodiments thereof, the foregoingdescription, as well as the examples which follow, are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

1. A method for forming an apparatus, said method comprising: providing first and second substrates and a seal; placing the second substrate on top of the seal placed on the first substrate so as to define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces, wherein: at least one of said inner surfaces is functionalized with polynucleotides, polypeptides, or polysaccharides; at least one of said first and second substrates comprises a means for nucleating a bubble; and at least one of said first and second substrates and said seal comprises an opening for introducing fluid into the closed chamber; and introducing a fluid into the closed chamber so as to provide a quantity of fluid therein in contact with both inner surfaces.
 2. A method according to claim 1 wherein the polynucleotide is a polyribonucleotide.
 3. A method according to claim 1 wherein the inner surfaces of the first and second substrates are substantially parallel.
 4. A method according to claim 1 wherein the chamber is up to three millimeters in thickness.
 5. A method according to claim 4 wherein the chamber is up to two millimeters in thickness.
 6. A method according to claim 1 wherein the seal is attached to the first substrate.
 7. A method according to claim 1 wherein the inner surface of the first substrate is functionalized with the polynucleotides, polypeptides, or the polysaccharides.
 8. A method according to claim 1 wherein the inner surface of the second substrate is functionalized with the polynucleotides, polypeptides, or the polysaccharides.
 9. A method according to claim 1 wherein the inner surfaces of the first and second substrate are each functionalized with polynucleotides, polypeptides, or the polysaccharides.
 10. A method according to claim 1 wherein one of the substrates has an opening for introducing fluid into the closed chamber.
 11. A method according to claim 1 wherein the seal has an opening for introducing fluid into the closed chamber.
 12. The method of claim 1 wherein the at least one of the inner surfaces is functionalized with polynucleotides.
 13. The method of claim 1 wherein the at least one of the inner surfaces is functionalized with polypeptides.
 14. The method according to claim 1, wherein said means for nucleating a bubble comprises discrete heat sources, discrete radiofrequency sources, discrete microwave sources, discrete light sources or discrete mechanical sources.
 15. The method according to claim 14, wherein said means for nucleating a bubble comprises discrete heat sources, wherein said discrete heat sources are resistors.
 16. The method according to claim 1, wherein said at least one of said inner surfaces functionalized with polynucleotides, polypeptides, or polysaccharides comprises known polynucleotides, polypeptides, or polysaccharides at specific locations.
 17. The apparatus according to claim 1, wherein said at least one of said inner surfaces functionalized with polynucleotides, polypeptides, or polysaccharides comprises known polynucleotides, polypeptides, or polysaccharides at specific locations.
 18. An apparatus comprising: first and second substrates and a seal, wherein the second substrate is positioned on top of the seal placed on the first substrate so as to define a closed chamber therebetween, said chamber retaining a quantity of fluid therein so that the fluid is in contact with both inner surfaces, and wherein: at least one of said inner surfaces is functionalized with polynucleotides, polypeptides, or polysaccharides; at least one of said first and second substrates comprises a means for nucleating a bubble; and at least one of said first and second substrates and said seal comprises an opening for introducing/fluid into the closed chamber.
 19. An apparatus according to claim 18 wherein the inner surfaces of the first and second substrates are substantially parallel.
 20. An apparatus according to claim 18 wherein the chamber is up to three millimeters in thickness.
 21. An apparatus according to claim 18 wherein the inner surface of the first and second substrate is each functionalized with polynucleotides, polypeptides, or the polysaccharides.
 22. An apparatus according to claim 18 wherein one of the substrates has an opening for introducing fluid into the closed chamber.
 23. An apparatus according to claim 18 wherein the seal has an opening for introducing fluid into the closed chamber. 